Fluorescence measurements are conventionally used in flow cytometry, fluorescence microscopy, and fluorescence spectroscopy. With few exceptions, such measurements are of the steady state emission signal, as intensity changes or wavelength shifts. Such measurements are often aimed towards determination of the concentration of fluorophore or analytes. The precise and accurate measurement of known intensity- or wavelength shift-based sensing has suffered from problems such as delayed results, the need for expensive, sophisticated and time consuming procedures that are limited, e.g., by high background noise, low signal-to-noise ratios, turbidity, optical losses, photobleaching and fluorophore washout; the need for alternative ranges of wavelength sensing; the need for continuous or immediate results; the need for containment of hazardous or potentially hazardous samples; the need for remote sensing; and/or the need for continuous or repetitive sampling.
These problems have the potential to be minimized or eliminated by the use of a measuring system based on fluorescence lifetime (or decay) measurements which are insensitive to changing instrument conditions and which are independent of the intensity fluctuations of fluorescence radiation. Fluorescence lifetime measurements can be performed in the time-domain by using the known time-correlated single photon counting (TCPSC) technique, or in the frequency-domain by measuring the phase shift and modulation of the fluorescence emission with respect to a amplitude-modulated sinusoidal excitation as a function of frequency.
In a TCPSC time-domain lifetime measurement system, the time dependent probability of fluorescence photon emission after pulsed excitation is measured by statistical counting of the arrival time of individual photons. However, the experimental data produced gives the fluorescence decay in a form that is convoluted with the instrument response function, and the use of time domain lifetime measurements are presently more expensive, time consuming and complex than frequency-domain measurements. See, e.g., "Pulsed Semiconductor Laser. Fluorometry for Lifetime Measurements", Analytical Chemistry, vol. 57, no. 4, April 1985, pp. 947-949.
In a conventional frequency-domain lifetime measurement system, a sample is excited with light from an intensity-modulated light source. Lifetime measurements are made by measuring the frequency response of the sample, i.e., the frequency-dependent phase angle and modulation of the fluorescence emitted from the sample. The emitted fluorescence is modulated at the same frequency as the excitation light but is shifted in phase and demodulated with respect to the excitation light.
For the case of pure sinusoidally modulated excitation and a single exponential decay, the phase shift .phi. and modulation ratio M of the fluorescence versus excitation are related to the excited state lifetime .tau. of the fluorophore and the modulation frequency .omega. by the following relations: EQU tan.phi.=.omega..tau. EQU M=M.sub.f /M.sub.r =(1+.omega..sup.2 .tau..sup.2).sup.-1/2
where M.sub.r and M.sub.f are respectively the excitation and fluorescence modulation amplitudes referenced to their respective DC levels. See, e.g., Lakowicz PRINCIPLES OF FLUORESCENCE SPECTROSCOPY Plenum Press, New York (1983). Also see, "AN AUTOMATED PHASE FLUOROMETER WITH HIGH PRECISION RESOLUTION FOR MULTI-EXPONENTIAL FLUORESCENCE DECAYS", by Clays, Engelborghs and Persons; REVIEW OF SCIENTIFIC INSTRUMENTS, 1988.
For a multi-exponential decay (wherein decays are present that are due to more than one fluorophore or analyte with differing lifetimes) the relations are EQU tan.phi.=S/G and M.sup.2 =S.sup.2 +G.sup.2
where S=.SIGMA..sub.i f.sub.i cos.phi..sub.i sin.phi..sub.i, and G=.SIGMA..sub.i f.sub.i cos.sup.2 .phi..sub.i, f.sub.i is the fraction of the total intensity from the ith component, and .phi..sub.i is the phase shift.
For a non-exponential decay, S and G should be calculated as the real and imaginary part of the Fourier transform of the response function I(t): ##EQU1## where I(t) is the time domain response function to a delta excitation function.
Suppose the excitation source produces a train of pulses, equally spaced in time, with constant repetition frequency f.sub.0 =1/T, constant shape (e.g., Gaussian), constant amplitude and width 2.DELTA., then the timing function of such a light source is given by ##EQU2## and the harmonic content in the power spectrum is given by its Fourier transform EQU G(f)=.delta.(f-nf.sub.0) exp (-1/2(.DELTA..f).sup.2)n=1,2,3, . . .
where .delta. is defined as the Dirac delta function, e.g., as described by K. Berndt, H. Durr and D. Kalme in Optics Communications, 1982, vol. 42, pp. 419-422.
If the pulse is short, the harmonic content in the power spectrum is high and energy is available over a wide range of frequencies. When excited with such a light source, the fluorescence of the sample will be modulated at all the higher harmonics of the pulse repetition frequency at the same time. Monitoring the phase shift at successive higher harmonics of the fundamental excitation frequency reveals information on the fluorescence decay. By measuring the phase shift at many harmonics in the power spectrum, the fluorescence lifetime resolution is increased, a feature which is particularly important for multi-exponential decays.
For example, the above described excitation source may be a synchronously pumped, cavity-dumped dye laser made by Spectra Physics (no. 375B), with Rhodamine 6 G (R6G) or 4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM) as the dye, pumped by the 514.5 nm green line of a mode locked Argon-ion laser (Spectra Physics 2030). Dye lasers of this type provide wavelengths ranging from 570 nm to 720 nm. Cavity dumping provides for more narrowly spaced harmonics at multiple frequencies, and frequency doubling of the laser output achieves excitation wavelengths in the ultraviolet (UV) frequency range, e.g. 285-340 nm. This UV output, used as an excitation light source, is useful for many fluorescence studies. The dye laser is equipped with a cavity dumper (Spectra Physics 344S) to reduce the pulse repetition frequency from the mode locked dye laser.
The pulsed laser source is thus advantageous in that the high peak power results in rather easy and efficient frequency doubling. The light output is intrinsically modulated over a wider range of frequencies, beyond that obtainable with any commercially available broadband modulator or other additional optical device that would otherwise be required. Using the harmonic content of the pulsed output of the dye laser as an excitation source considerably improves the accuracy and resolution of phase measurements. In this manner, phase shift data can be read out within a relatively short measurement time. All the harmonics within the bandwidth of the system are of comparable amplitude because of the very high bandwidth of the excitation function.
However, the above described laser system is extremely expensive, requires considerable space and power, and is difficult to align and maintain. Thus, there is a need for a fluorescence lifetime measuring apparatus and method which provide improved accuracy, shorter measurement times and lower power requirements, without the high cost and maintenance problems of the prior art systems.